Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

RationaleProtein dynamics are fundamental for biological activity. Direct surface functionalization with these biomolecules often creates regions presenting non-homogeneous and altered protein functionality. Aptamers have been shown to be an effective method to enhance the biocompatibility of biomaterials by acting as docking points for biomolecules capable of improving cell responses and the colonization/proliferation processes. Here mass spectrometry (MS) was successfully applied as an analytical approach to study the interactions between a fibronectin fragment (FN) and anti-fibronectin aptamers in solution and on a chitosan film.MethodsA 30kDa N-terminal fibronectin fragment, a ssDNA anti-fibronectin 40 nucleotide long aptamer and chitosan 95/50 (degree of deacetylation 92.6-97.5%; molecular weight 80-220kDa) were used. First, the dynamics of the FN in aqueous solution, in the presence or absence of anti-FN-specific DNA aptamers, were described. Thus, the same study was carried out on 2% (w/w) chitosan-based films, functionalized with FN alone or through aptamers as selective spacers.ResultsAmide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis identified the SYRIGDTWSKKDNRGNL and YRVGDTYERPKDSMI, YVVGETWEKPYQGWMM and WERTYLGNAL fragments as presumably involved in the interaction of FN with aptamers. MS findings indicated the improved functionality of FN on chitosan when aptamers were used as selective spacer, and this may explain why cells preferentially attached to aptamer-bound FN rather than on chitosan-FN films.ConclusionsAptamers did not affect the amount of adsorbed FN, but influenced its conformation enhancing its biological activity toward adhesion and growth of osteoblasts. HDX-MS data allowed the identification of the FN/aptamer interaction regions. Differences in FN flexibility on the chitosan film in the presence or absence of the aptamer were established providing insights at the molecular level to better understand the protein biological functionality on cell proliferation.